In a clinical test or the like, highly sensitive and quantitative detection of a trace amount of analyte, such as a protein or DNA, would allow a quick understanding of a patient's condition and the subsequent his/her treatment. For this reason, there is a need for a method of detecting a trace amount of analyte highly sensitively and quantitatively.
As a highly sensitive method of detecting an analyte, surface plasmon-field enhanced fluorescence spectroscopy (hereinafter abbreviated as “SPFS”) is known. SPFS utilizes surface plasmon resonance (hereinafter abbreviated as “SPR”) generated by irradiating a metal film with light under specific conditions (see, Patent Literature (hereinafter abbreviated as PTL) 1, for example).
In SPFS, a ligand (e.g., primary antibody) that can specifically bind to an analyte is first immobilized above a metal film, thereby forming a reaction site for specifically capturing an analyte. When a sample containing an analyte is provided to the reaction site, the analyte binds to the ligand in the reaction site. Then, when another ligand (e.g., secondary antibody) labeled with a fluorescent substance is provided to the reaction site, the analyte bound to the ligand in the reaction site is labeled with the fluorescent substance. When the metal film is irradiated with excitation light in this state, the fluorescent substance that labels the analyte is excited by enhanced electric fields due to SPR to emit fluorescence. Thus, the detection of emitted fluorescence allows the detection of the presence or an amount of the analyte. SPFS can detect an analyte highly sensitively since a fluorescent substance is excited by enhanced electric fields due to SPR.
During the detection of fluorescence, however, the presence of an unreacted fluorescent substance that does not label an analyte above a metal film results in background noise. Accordingly, in order to detect an analyte accurately, it is preferable to remove an unreacted fluorescent substance in advance by washing.
SPFS is broadly categorized into prism coupling (PC)-SPFS and grating coupling (GC)-SPFS in accordance with a means for coupling excitation light with surface plasmon. PC-SPFS utilizes a prism formed on one surface of a metal film. In this method, excitation light and surface plasmon are coupled by total reflection of excitation light at an interface between the prism and the metal film. Although PC-SPFS is a mainstream method today, it has a challenge in downsizing a detection apparatus since a prism is used and an incident angle of excitation light on a metal film is large.
In contrast, GC-SPFS couples excitation light with surface plasmon utilizing a diffraction grating (see PTL 2, for example). GC-SPFS can downsize a detection apparatus compared with PC-SPFS, since a prism is not used and an incident angle of excitation light on a diffraction grating is small.